![]() ![]() The final vRNA was transferred in 15 μl and 8 μl volumes to two 0.2 ml PCR tubes, (cat. The final elution volume of 50 μl was supplemented with RNase Inhibitor (0.8 units/μl Life Technologies). #R1018, Zymo Research Irvine, CA) as per the manufacture’s protocol. To further improve vRNA recovery, the eluted vRNA (100 μl) samples were subjected to a second RNA extraction/elution using the RNA Clean and Concentrator kit – 25 (cat. The increased elution volume allowed for higher vRNA recovery from the supplied spin filter tubes containing glass fiber fleece. #11858882001, Roche Indianapolis, IN) using the manufacture’s protocol with the modification that the elution volume was increased from 50 μl to 100 μl and supplemented with RNase Inhibitor (0.3 units/μl cat. Viral RNA (vRNA) was extracted from plasma using High Pure Viral RNA kit (cat. #AM2548, Life Technologies Carlsbad, CA) was added to neat plasma or virus pellet specimens at a final concentration of 2.5 mg/ml followed by incubation at 37☌ for 30 min to inactivate further proteolysis. To inactivate nucleases and other contaminants, proteinase K (cat. The centrifugation step allows for quantification of virus from higher volumes of plasma when PVL is expected to be low. After centrifugation, the excess plasma supernatant was carefully removed from the virus pellet before continuing with the vRNA extraction protocol. Plasma samples were assayed neat or centrifuged at 25,000 × g for 1 hour to pellet virus particles. For each assay, 10 ul aRNA were added to each plasma sample, and based on the manufacturer’s information, the copy numbers fell within the dynamic range of the HCV-2b calibration curve. coli bacteriophage MS2 protein coat was used as the IPC to monitor RNA recovery and quantification. #42010, Asuragen Austin, TX) containing a short RNA sequence packaged in an E. ![]() In selected experiments where indicated, armored RNA (aRNA) of Hepatitis C Virus genotype 2b (HCV-2b cat. This study was performed and results presented in compliance with the “ minimum information for publication of quantitative real-time PCR experiments” guidelines (MIQE ). The results from the qPCR were correlated with the branched-DNA (bDNA) signal amplification and hybridization. The aRNA for Hepatitis C Virus (HCV) was chosen as the IPC because HCV does not infect NHPs to confound data interpretation. Our overall approach was to optimize SIV PVL qPCR assay for (SIVmac239, SIVsmE660 and SIVagmSAB) using aRNA and an internal positive control (IPC). To date however, aRNA has not been used for quality control in quantification of SIV RNA. ![]() Commercially-available aRNAs have been used in human diagnostics for quality assurance in viral load determination, including human immunodeficiency virus (HIV). ![]() Known concentrations of aRNA can be spiked into test specimens to control for yields in sample preparation and reverse transcription. Armored RNA (aRNA) contains a short RNA viral sequence packaged in an Escherichia-coli bacteriophage MS2 protein coat to represent a synthetic target virus. The purpose of this study was to improve sensitivity and reproducibility of SIV PVL qPCR through optimizing various multiple steps of SIV PVL qPCR (sample preparation, cDNA synthesis, and amplification) and by using heterologous armored RNA (aRNA). Inadequate preparation of plasma, suboptimal vRNA extraction efficiency, and poor primer and probe design also each contribute to inaccurate quantification of viral copy number in plasma. Plasma is a challenging specimen for quantification of viral RNA due to the presence of PCR inhibitors and RNAses, and removing such inhibitors improved analytical sensitivity for detection of viral RNA. Quantitative polymerase chain reaction (qPCR) is commonly used for determination of PVL. Plasma viral load (PVL) is widely used to assess the status and progression of simian immunodeficiency virus (SIV) infections in nonhuman primates (NHP). ![]()
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